Cell transplantation shows potential promise for treating ischemic heart disease. Several elegant studies have demonstrated that implantation of skeletal myoblasts and bone marrow stem cells into the scarred myocardium can result in improved function. However, longitudinal analysis of cell survival remains problematic, because the current methodology relies on postmortem histology. Recently, we have established the feasibility of monitoring transplanted embryonic cardiomyoblasts expressing reporter genes in living animals using optical bioluminescence charged coupled device (CCD) and micro positron emission tomography (microPET) imaging. The specific aims of this proposal are well- thought-out extensions of our initial findings. This proposal will test the following multi-leveled hypothesis. In Aim 1, we will continue to develop an in vivo imaging model for monitoring the location, magnitude, and survival duration of transplanted human mesenchymal stem cells (hMSCs). Equally important, cell survival will be correlated with changes in contractility, perfusion, and metabolism, as determined by echocardiography, [13N]-NH3, and [18F]-FDG imaging, respectively. In Aim 2, imaging results will be validated with traditional ex vivo and in vitro assays, such as morphometric analysis, immunocytochemistry, TaqMan PCR, TUNEL apoptosis stain, Westerns, and enzyme assays. Afterwards, we will focus on more fundamental questions related to stem cell physiology. In particular, Aim 3 will evaluate the optimal cell type, cell dosage, delivery route, timing of transplant, and efficacy of repeated injections. Finally, we will examine the causes of early cell death post transplant in Aim 4, and then screen for pharmaceutical, gene, and cell-based therapies aimed at preventing cell death and apoptosis. The overall significance of this proposal is to understand the real-time physiologic state of transplanted cells in an intact whole-body system. These studies will add further insights to the field of stem cell biology. As PET imaging is readily transferable from small animals to humans, our findings should have direct clinical impact in the near future. The goal is to use molecular imaging to guide stem cell therapy, which should translate into transplant protocols that are more reproducible, quantifiable, and beneficial.